In this study, 2′-FL significantly reduced oxidative stress damage and inflammation in the intestines of aging mice, potentially by regulating the sirtuin1 (SIRT1)-related and nuclear factor E2-related factor 2 (Nrf2) pathways. In addition, 2′-FL significantly improved the gut mucosal barrier function and increased the content of short-chain fatty acids (SCFAs) in the intestine. The gut microbiota analysis indicated that 2′-FL mainly increased the abundance of probiotics like Akkermansia in aging mice. Moreover, 2′-FL significantly inhibited apoptosis in the brains of aging mice, also increasing the expression of SIRT1. These findings provided a basis for learning the benefits of 2′-FL in the aging process.The imbalance of reactive oxygen species is the main cause in aging, accompanied by oxidative stress. As the most abundant in human milk oligosaccharides (HMOs), 2′-Fucosyllactose (2′-FL) has been confirmed to have great properties in immunity regulation and anti-inflammatory. The research on 2′-FL is focused on infants currently, while there is no related report of 2′-FL for the elderly. A  SIRT1 is involved in DNA repair, apoptosis, inflammation, aging, and other biological processes [Aging is a complex physiological process, leading to a gradual decline in physical health . Under nrocesses . Nuclearrocesses . The actrocesses .Firmicutes/Bacteroides [Escherichia coli by lowering the pH, and stimulate the growth of beneficial bacteria of Firmicutes [Aging is closely related to changes in the environment of the intestine. A variety of bacteria are colonized in the intestine to form the gut microbiota. Aging decreases the diversity of gut microbes, like butyrate-producing species, and changes the composition, like a decrease in the abundance of teroides ,12. Shorteroides ,14. The rmicutes ,16. In armicutes . Aging crmicutes .Aging usually affects the physiological functions of the brain, accompanied by various neurodegenerative diseases like Alzheimer’s disease. Studies have reported that excessive apoptosis activation is increasingly involved in several neurodegenerative diseases related to aging . MoreoveBifidobacterium, inhibiting the reproduction of harmful bacteria [Streptococcus mutans found that GOS promoted the growth of Streptococcus mutans as a carbon source, while 2′-FL could not. Both could reduce the adhesion of pathogenic bacteria, but the mode of inhibition might be different [E. coli O157 in the intestines of mice was decreased by over 90%. One of the potential reasons was that 2′-FL is a structural analog of intestinal mucosal glycoprotein, which acts as a bait receptor against pathogenic bacteria. [As a natural prebiotic, 2′-fucosyllactose (2′-FL) is the trisaccharide with the highest content in human milk oligosaccharides (HMOs), accounting for 31% and constituting glucose, galactose, and fucose . 2′-FL ibacteria ,24, regubacteria . At presbacteria . Chitosabacteria . In addibacteria . It is wbacteria . Anotherifferent . Previouacteria. . In a poacteria. .d-galactose  into mice were first reported in China for signs of accelerated aging. Neurological damage, decreased antioxidant enzyme activity, and a weakened immune response have been demonstrated in this model of aging. d-gal is a reducing sugar that reacts with free amino groups of protein and peptide amino acids to form advanced glycation end products. These biochemical processes mentioned cause oxidative stress and cellular damage. d-gal is converted to galactitol, which can produce reactive oxygen species in the cell and stimulate osmotic stress, as well as accumulate in the cell. This study will apply the d-galactose-induced accelerated aging model to investigate the potential mechanism of 2′-FL on the intestine and the effect on the repair of brain damage in aging.As a natural ingredient in breast milk, the research on 2′-FL is focused on infants currently. However, there is no related report of 2′-FL for the elderly. Based on the function of 2′-FL, we reasonably speculate that 2′-FL intervention can work potentially in treatment strategies for aging. Injections of low-dose d-gal was used to build an accelerated aging model. Long-term injection of excessive d-gal could cause oxidative stress damage and inflammation, which led to aging and nerve dysfunction. This model was usually used for research on aging-related aspects.Male-specific pathogen-free (SPF) ICR mice (10 weeks old) purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd.  were raised and tested in accordance with the institutional guidelines. d-gal model group , and 2′-FL intervention group (2′-FL). All animals were housed individually at 22–23 °C, 46–53% relative humidity with 12 h:12 h light–dark cycles. In the d-gal model and 2′-FL intervention groups, mice were injected with 300-mg/kg/day d-gal. Mice in the control group was injected with 0.9% saline with equal volume, relatively. The 2′-FL intervention was added to the feed at a 2.5-g/kg diet in the 2′-FL intervention group. The specific diet composition is listed in In our study, mice were adapted to the environment with a standard diet for 1 week and then divided into 3 groups (12 mice in each group) that were the control group (Control), This animal experiment was ethically reviewed by the Institutional Animal Care and Use Committee of Nankai University, and the ethical approval number is 2021-SYDWLL-000472. All experimental procedures followed the guidelines of international animal welfare and ethics.Blood samples stored at −80 °C were used to measure oxidative stress indicators. Biochemical indexes were measured with commercial detection biochemical kits , including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) in the serum.qPCR was applied to determine the expression levels of genes in the colon and brain tissues. In the colon, the genes intestinal barrier-related, including mucoprotein 2 (MUC2), Claudin1, and E-cadherin; the genes inflammation-related, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β); the genes oxidative stress-related, including Nrf2, Kelch-like ECH-associated protein 1 (Keap1), HO-1, and NADPH quinone oxidoreductase-1 (NQO-1); the genes of SCFAs receptors, including G-protein-coupled receptor 41 and 43 (GPR41 and GPR43); and the genes aging-related, including SIRT1, FOXO1, and PGC-1α, expression levels were determined by qPCR. In the brain, the genes apoptosis-related, including B-cell lymphoma-2 (Bcl-2), Bax, and caspase-3; the genes oxidative stress-related, including Nrf2, Keap1, HO-1, and NQO-1; and the genes aging-related, including SIRT1 and FOXO1, were measured for their expression levels by qPCR. The experimental methods are shown in the Western blotting was applied to determine the expression levels of proteins in the colon. We measured the protein expression levels of SIRT1, FOXO1, p-AMPK, and AMPK in the colon. The specific methods are shown in the 2O2 soaking for 10 min. Then, a citric acid buffer was added to the sections and put in a microwave oven for 3 min. The serum was used to block the sections at 37 °C for half an hour and then incubated overnight at 4 °C with the primary antibody . The mixture was incubated at 37 °C for half an hour with the secondary antibody , followed by adding developers to develop the color. The color-developed film was rinsed with water for a while and later soaked in hematoxylin for half a minute. The counter-stained film was dehydrated and covered with a cover glass, sealed, and dried for microscopy.The intestinal tissue was made into paraffin sections with a thickness of 5 µm and stained for the expression of MUC2 in the mucosal layer. Immunohistochemistry was performed by the Servicebio Company , and the method was as follows: The sections were deparaffinized for 10 min, followed by 3% HThe brain tissue was made into paraffin sections about 5 μm, which were stained with H&E, encapsulated as slides, and photographed under the microscope for analysis.The fecal sample was lyophilized, its dry weight weighed, homogenized in pure water (1 mL), and then centrifuged  to obtain the supernatant. The supernatant was added to 100 μL of 10% sulfuric acid and 0.5 mL of ether and vortexed for 2 min. Continuously, the mixture was extracted for 5 min under the shock. The extracted solution was centrifuged  to take the above ether phase through 0.22-μm filter membranes. Finally, the ether extract was stored in a brown sample vial to determine the SCFA content. A DB-FFAP capillary column  was used for determination in gas chromatography . Agilent’s MSD ChemStation (E.02.00.493) was adopted for data processing.Bacterial DNA extraction, PCR amplicon sequencing, and genetic analysis in the cecum content were carried out by the Genedenovo Biotechnology Company . In order to analyze the gut microbiome in the cecum content, we used 16S sequencing technology. The V3 + V4 domains of 16S rDNA were amplified by barcoded specific primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTATTCTAAT). Qubit 3.0 fluorometer was used for recovery and quantitative amplification, and a sequencing library was constructed according to the official Illumina information. Finally, sequencing was completed using the PE250 model in the Hiseq2500 system.p < 0.05. Letters a, b, and c were applied in all histograms, indicating significant differences between groups. A gut microbiota analysis was conducted using Omicsmart with the online platform (http://www.omicsmart.com accessed on 13 December 2021).Statistical analysis was performed using Origin software . Data were normally presented as the mean ± standard error of the mean (SEM). Difference analyses of the results were conducted by the one-way ANOVA test with Fisher LSD multiple comparisons, while statistical significance was determined as d-gal-induced aging model. As shown in p < 0.05), and MDA was significantly increased (p < 0.05) in the d-gal group compared with the control group. The dietary intervention of 2′-FL increased the level of antioxidant enzymes in the blood (p < 0.05), reducing the oxygen free radicals produced by the enzyme system and improving the antioxidant capacity. Otherwise, MDA was a product of lipid peroxidation induced by oxygen free radicals, promoting lipid oxidation and forming a vicious cycle. The 2′-FL intervention significantly reduced the production of MDA in the blood (p < 0.05).The levels of SOD, CAT, GSH, GSH-PX, and MDA in the serum were measured for the effects of 2′-FL in the d-gal group was lowest among the groups, with extremely scarce mucin in the goblet cells. All of them indicated that aging was positively associated with the decline of the gut mucosal barrier function. Furthermore, the gut mucosal barrier-related genes expression was determined to measure the defense functions. In the d-gal group, the expression level of MUC2, Claudin-1, and E-cadherin significantly decreased (p < 0.05) compared with the control group. The expression level of the gut mucosal barrier-related genes in the 2′-FL intervention group increased compared with the d-gal group (p < 0.05), but there was still a gap with the control group level  compared with the control group. When intervened by 2′-FL, the expression level of TNF-α and IL-1β significantly decreased (p < 0.05). The expression of IL-1β had no difference between the control group and the 2′-FL intervention group, but TNF-α was still a little higher than the control group. These data indicated that the 2′-FL intervention effectively reduced the inflammatory response in the d-gal model.The relative mRNA expression of inflammatory cytokines in the colon was measured to evaluate the effect of the 2′-FL intervention on inflammation. As shown in d-gal treatment significantly decreased the expression levels of Nrf2, NQO1, and HO-1 (p < 0.05) but increased the expression level of Keap1 (p < 0.05). However, 2′-FL could reverse this situation; the relative gene expression levels of Nrf2, NQO1, and HO-1 were all significantly increased (p < 0.05), while the level of Keap1 was significantly decreased (p < 0.05)  b.d-gal group were significantly reduced (p < 0.05). Compared with the d-gal group, the 2′-FL intervention group significantly upregulated the expression levels of these genes (p < 0.05), which were not significantly different from the control group. Furthermore, the expression of AMPK, pAMPK, SIRT1, and FOXO1 were determined at the protein level. These results showed that the protein levels of pAMPK, SIRT1, and FOXO1 in the 2′-FL intervention group were significantly upregulated compared with the d-gal group, but there was no significant difference from the control group on pAMPK and SIRT1. These data indicate that the 2′-FL intervention has effects on inhibiting the process of aging through the AMPK/SIRT1/FOXO1 signaling pathway.qRT-PCR and Western blotting were applied to determine the AMPK/SIRT1/FOXO1 longevity-related signaling pathway in the colon for the mechanism of 2′-FL intervention on aging. As shown in d-gal group, although the expression level of GPR41 increased, there was no significant difference. The GPR43 expression level of the 2′-FL intervention group was significantly upregulated compared to the d-gal group (p < 0.05) but not different from the control. To verify the changes in the GPRs, we further measured the content of SCFAs in the colon contents. We found that the contents of acetic acid, propionic acid, and butyric acid in the 2′-FL intervention group were highest among the groups (p < 0.05), while the content of valeric acid was not different in all the groups (The AMPK pathway could be activated by G-protein-coupled receptors (GPRs). We believe that 2′-FL may bind to receptor members of the GPR family through its metabolite SCFAs, thereby activating the downstream AMPK signaling pathway. We measured the expression of GPR41 and GPR43, which were main receptors of SCFAs in the colon. As shown in e groups c. These Firmicutes and Deferribacteres in the d-gal treatment group increased, but the relative abundance of Verrucomicrobia decreased. After a long-term 2′-FL intervention, the relative abundance of Verrucomicrobia reversed dramatically compared with d-gal. The proportion of Verrucomicrobia was 0.89% in the d-gal treatment group and 3.5% in the 2′-FL intervention group. In addition, the ratio of Firmicutes/Bacteroidetes also increased significantly , while the relative abundances of Ruminiclostridium and Ruminiclostridium_9 were extremely significantly increased (p < 0.01). Surprisingly, the 2′-FL intervention significantly reversed the changes. The relative abundance of Akkermansia in the 2′-FL intervention group significantly increased and restored its original abundance, and the relative abundance of Ruminiclostridium_9 and Butyricimonas was significantly reduced (p < 0.05) , while the expression of cancer-related pathways was significantly upregulated (p < 0.05). The 2′-FL intervention group significantly increased the expression of translation function (p < 0.05) and decreased the expression of neurodegenerative disease-related pathways compared with the d-gal treatment group (p < 0.01).16S sequencing technology was applied to measure the species differences of the gut microbiota on 2′-FL intervention. At the phylum level, the relative abundance of ficantly a. At the < 0.05) b. The diAkkermansia was positively correlated with the expression of longevity genes SIRT1 and FOXO1 but negatively correlated with IL-1β. Some unidentified bacteria such as Ruminiclostridium and Ruminiclostridium_9 were negatively correlated with the expression of gut mucosal barrier-related genes  but positively correlated with inflammatory cytokines (TNF-α). At the species level . In addition, Lachnospiraceae_bacterium_DW17 positively correlated with inflammatory cytokines (TNF-α and IL-1β). These data supported the conclusions that the interaction of 2′-FL with the gut microbiota had a positive effect, increasing the abundance of beneficial bacteria like Akkermansia and regulating the expression of genes related to inflammation and antiaging.Moreover, the Pearson correlation analysis was used to investigate the relevance level between the gut microbiota and environmental factors . At the genus level d, Akkermes level f, Akkermd-gal. In addition, we measured the expression levels of apoptosis-related genes in the brain. As shown in d-gal group (p < 0.05). The expression level of Bcl-2 in the 2′-FL intervention group was the highest among the groups (p < 0.05).Aging would reduce cone cells and inflammatory infiltration in the hippocampus of the host’s brain, thereby promoting the production of inflammatory cytokines and oxidative stress products, ultimately leading to a series of degenerative changes such as neurotransmitter metabolism disorders. As shown in p < 0.05) but was not different from the control group in the SIRT1 level , which showed that d-gal had little effect on the Nrf2 signaling pathway in the brain. Compared with the d-gal group, the 2′-FL intervention group significantly increased (p < 0.05) the levels of Nrf2 and NQO-1 , leading to tissue damage and cell apoptosis . IL-1β iE. coli O157 infection model in vivo, the 2′-FL intervention significantly increased the expression levels of MUC2 and tight junction protein occludin [The intestinal barrier integrity can be destroyed by inflammation during the aging process . In the occludin . Moreoveoccludin . These soccludin . In thisFirmicutes/Bacteroides and Akkermansia in the intestine decreases, whereas the abundance of those harmful bacteria such as Proteus, Enterobacter, and Enterococcus increases [Akkermansia in thin people. This has led to claims that this bacterium can prevent weight gain. One of the foods that will feed the bacteria is red grapes. During the aging process, these changes in the gut microbiota can cause a series of diseases. In this study, the 2′-FL intervention significantly increased the abundance of Verrucomicrobia, while Akkermansia contributed the most at the genus level. Furthermore, the correlation analysis of the environmental factors proved that Akkermansia is positively correlated with the SIRT1-related longevity signaling pathway. Akkermansia is one of the few known species in the Verrucomicrobia phylum that can degrade mucin into acetic and propionic acids, further promoting intestinal integrity [Akkermansia was found to extend the lifespan of premature aging mice. Moreover, in a cohort study, it was also found that Verrucobacteria in the gut microbiota of centenarians increased, while the proteobacteria decreased, indicating that Akkermansia had the potential to extend the lifespan [Butyricimonas in the 2′-FL intervention group was decreased. However, there are reports that one species called Butyricimonas virosa under Butyricimonas is a harmful bacterium that can cause bacteremia [Lachnospiraceae_bacterium_615 and Lachnospiraceae_bacterium_DW17 under Lachnospiraceae have a negative correlation with the intestinal barrier. Although members of the Laospirillaceae family are the main producers of SCFAs, the different taxa of the Laospirillaceae are also associated with different enteral and extraintestinal diseases. Specific taxa of the Laospirillaceae family were involved in metabolic syndrome, obesity, diabetes, liver disease, and inflammatory bowel disease [The dynamic balance of the gastrointestinal tract is generally maintained by the gut microbiota. The composition of the gut microbiota, as well as abundance, would change differently during different life periods. With the increase of age, the abundance of ncreases . It has ntegrity . In a stlifespan . In addicteremia . It is w disease . These o+-dependent deacetylase, SIRT1 can regulate the AMPK signaling pathway through the deacetylation of certain key proteins , s PGC-1α ,60,61. IIn the colon, 2′-FL is metabolized by bacteria into a variety of SCFAs . SCFAs c3-induced cognitive impairment in rats, a lycopene intervention was considered to inhibit the process of apoptosis, which was accompanied by a weakness of the hippocampal lesions [d-gal.Aging is always accompanied by the deterioration of brain function, such as oxidative stress damage, cognitive impairment, increased apoptosis, and neurodegenerative diseases. In a study of AlCl lesions . Bcl-2 i lesions . The cas lesions . As mentd-gal-induced aging model. Its mechanism depended on the regulation of the AMPK/SIRT1/FOXO1 and Nrf2/Keap1 signaling pathways, respectively. 2′-FL had a relieving effect on brain damage, effectively inhibiting the apoptosis of brain tissue cells. These results indicated that 2′-FL and its metabolites had a regulatory effect on the homeostasis of the gut environment and a potential protective effect on the brain. This study clarified that 2′-FL supplementation was beneficial in the aging process, providing a new idea for comprehending the function of 2′-FL.In conclusion, 2′-FL was beneficial in oxidative stress, the gut mucosal barrier, and inflammation in the


Keratoconus is a progressive corneal disease commonly treated by collagen cross-linking (CXL). Accelerated protocols have recently become common. This study sought to compare the outcomes of accelerated and standard CXL in terms of visual acuity, keratometry, and tomographic parameters in pediatric population. Kmax, Ksteep, Kflat, Kmean), and endothelial density count (EDC) were assessed before and at 6 and 12 months following treatment. The analysis included intergroup and intragroup comparisons.  We retrospectively reviewed the files of pediatric patients who underwent standard and accelerated CXL for keratoconus in our hospital, between October 2014 and March 2018. Changes in uncorrected distance visual acuity (UCDVA), best corrected distance visual acuity (BCDVA), tomographic keratometry parameters , while 39 underwent accelerated CXL . Intergroup comparison found insignificant differences between groups, with the exception of better results for UCDVA in the S-CXL group after 12 months (P = 0.03). In this study, there was no significant difference between the two protocols postoperatively in BCDVA, Kmax, Kmean, pachymetry, or corneal astigmatism.  This study included 53 eyes (44 patients). Fourteen eyes were treated with standard CXL (S-CXL, 3 mW/cm A-CXL is as safe and effective as S-CXL for stabilizing progressive keratoconus in pediatric population. Larger-sample-size studies with a longer follow-up time are required. Considering the long-term results of 9 mW A-CXL and its safety and efficacy profile, it should be preferred to S-CXL for reducing treatment time and improving patients' comfort. Keratoconus is an ectatic disease of the cornea leading to visual impairment . ClassicWollensak et al. were the first to report the standard cross-linking protocol  in 2003 . S-CXL, An accelerated protocol for cross-linking (A-CXL) has been recently become common. It is based on the Bunsen–Roscoe law of reciprocity, which allows treatment time to be shortened by increasing the radiation intensity to deliver the same total energy dose. Shorter treatment time is especially important in children, because it can improve their compliance with treatment , 14.Studies on adults have shown similar outcomes in different corneal parameters between the S-CXL and the A-CXL protocols , 16. In In the current retrospective study, we compared visual acuity, keratometry, and tomographic criteria between the S-CXL and A-CXL protocols in children with keratoconus at 6 and 12 months following the procedure, in order to determine if the A-CXL is as safe and efficient as the S-CXL.Kmax or a decrease of 5% in central corneal thickness at two consecutive evaluations with Pentacam. Patients with progressive keratoconus were treated with either the S-CXL or A-CXL regimen. All patients were treated with CXL after instillation with isotonic riboflavin. Clinical examination and tomography were performed before and 6 and 12 months after CXL treatment.We retrospectively reviewed the files of pediatric patients who underwent S-CXL and A-CXL for keratoconus at the Department of Ophthalmology in Soroka University Medical Center, between October 2014 and March 2018. The diagnosis of keratoconus and its progression was made using a Pentacam tomographer . The preoperative progression was defined as a 1.5 D increase in the mean keratometric value or 1 D increase in Kmax over 68 D, central corneal thickness less than 400 μm, history of recurrent corneal erosion or dystrophies, history of corneal herpes virus infection, history of rheumatological and autoimmune disease, or sensitivity to any of the substance that is used in the CXL procedure.We included patients aged 18 years and below diagnosed with progressive keratoconus and treated with CXL who completed 12 months of follow-up after the procedure. We excluded patients with a history of any ocular disease or surgery, 2 for 30 min (S-CXL) or 9 mW/cm2 for 10 min (A-CXL). Riboflavin solution was instilled continuously every 2 minutes during UV-A irradiation. The patient was instructed to fixate on the light source, and adequate centration was constantly monitored by the surgeon. All eyes were bandaged immediately after the procedure.CXL was performed under topical anesthesia, with oxybuprocaine hydrochloride 0.4% eye drops used before the procedure. A 8.0 mm diameter of the central corneal epithelium was removed using a blunt spatula or epithelial peeler. Then, iso-osmolar riboflavin  was instilled after epithelial removal every 2 minutes for 30 minutes based on the Dresden protocol. UV-A was then irradiated at an intensity of 3 mW/cmFollow-up visits were routinely performed at 1 day, 7 days, 1 month, 6 months, and 12 months following CXL. All patients were prescribed topical ofloxacin 0.3% qid for 10 days and topical dexamethasone 0.1% qid for a total of one month, with gradual tapering down. Patients were advised to use preservative-free artificial tears as needed. One day following the procedure, a therapeutic contact lens was placed if corneal erosion was greater than 3 × 3 mm . The contact lens was removed during the 7-day follow-up. Patients underwent corneal tomography using Pentacam and EDC using Specular (noncontact specular microscopy) both preoperatively and at the 12-month follow-up.The study protocol was approved by the Institutional Review Board of “Soroka University Medical Center,” Beer-Sheva, Israel, and adhered to the tenets of the Declaration of Helsinki. Data regarding patients with keratoconus were retrospectively collected from medical reports in outpatient clinics. Keratometry and tomographic parameters were acquired using a rotating Scheimpflug tomography camera . EDC was measured with automated specular microscopy . Visual acuity parameters were collected using a Snellen chart and converted into logMAR for statistical analysis. BCDVA parameters were collected for patients that use glasses or contact lens and were fitted after the CXL procedure. Demographic parameters  were extracted from patient files.Kmax progression (defined as an increase of 1 D or more in Kmax) and regression (defined as a decrease of 1–D or more in Kmax), at 12 months following the procedure. The secondary outcome measures included UCDVA, BCDVA, corneal tomographic parameters, minimum corneal thickness at the last visit, EDC values, and postoperative keratoconus.The primary outcome measure in this study was T test. The categorical characteristics were described as frequency and percent and analyzed using a chi-square test. In addition, for the primary outcome, we compared between the groups the frequency of patients with Kmax < 1 D (improvement) and patients with Kmax >1 D (progression), at 6 and 12 months after treatment using a chi-square test. A P value < 0.05 was considered significant, and all analyses were conducted using SPSS version 24.First, we described the demographic differences between the two groups. Age was described by the mean and standard deviation, and the comparison was performed by the T tests with a threshold for significance at α = 0.05. In addition, to assess either an improvement or worsening following the intervention, we compared the measured parameters before and at multiple points after intervention within each study group (intragroup), before versus 6 months after treatment and before versus 12 months after treatment, using a paired T test with a threshold for significance at α = 0.05.To assess differences between the study groups, we compared the eye characteristic changes (deltas) between them (intergroup). Results were described by the mean and standard deviation and analyzed by the unpaired The study included 53 eyes of 44 patients. Fourteen eyes (13 patients) received S-CXL treatment (26%), while 39 eyes (34 patients) received A-CXL treatment (74%). The mean age of the patients was 15.4 years in the S-CXL group and 14.9 years in the A-CXL group. Fifty percent of the S-CXL group and 59% of the A-CXL group were males. Patients' demographics and clinical characteristics are shown in Kmax was observed after 12 months in 7 (18.4%) eyes of the A-CXL group and in 1 (7.1%) eye of the standard CXL group  eyes of the A-CXL group and in 7 (50%) eyes of the S-CXL group  and K1 front after 6 months .P = 0.04). There was only insignificant improvement in mean UCDVA after 12 months, with better results in the S-CXL group. There was no significant difference from baseline in Kmean, Ksteep, and Kflat for either group. There was also no significant difference from baseline in Kmax, but we found a progressive improvement trend in the S-CXL group (reduction of 0.8 D after 1 year), compared to slight elevation (0.2 D) after 6 months and stabilization after 12 months in the A-CXL group. The mean EDC was insignificantly decreased in both groups at 6 and 12 months following the procedure , which was significantly better in the S-CXL group at the 12-month follow-up. Moreover, in most measured parameters, the use of S-CXL showed better keratometric and visual acuity outcomes than of the A-CXL procedure, but these differences were not significant.2 protocol in a prospective nonrandomized cohort of 156 eyes of 112 patients with keratoconus, including 40 eyes of pediatric patients. The study demonstrated statistically significant improvements in UCDVA, BCDVA, Kmax, and corneal higher-order aberrations that lasted until the end of follow-up time. Mazzotta et al., in this important report, concluded finally that the 9 mW 5.4 J/cm2 A-CXL is the natural evolution of Epi-Off CXL treatment for the management of early progressive corneal ectasia and thus optimizes clinic workflow [2) resulted in no disease progression after 24 months [Several A-CXL protocols have been found to be safe and efficient, similar to this study. Mazzotta et al. in the recently published “Siena Eye-Cross Study 2” reported long-term results of 5 years of the A-CXL Epi-Off 9 mW/5.4 J/cmworkflow . Moreoveworkflow –22. Shetworkflow , while O4 months .2 for 10 minutes, with a follow-up time of 1 year. The results showed no significant difference between groups [2) with a 36-month follow-up and found a significantly better result in the A-CXL group after 1 year and 36 months in terms of visual acuity, Kmax, and manifest refractive spherical equivalent. Notably, while this study compared mean values, the current study compared mean changes [However, only few studies have compared both methods in the pediatric population, with differing results –26. Baenn groups . Sarac en groups . The thiKmax value of the groups, the primary outcome, similar to the findings of Baenninger et al. and Sarac et al. In contrast, Eissa et al. found a significant difference between the groups in this parameter with better results in the A-CXL group [In this study, there was no significant difference between XL group –26.Regarding visual acuity, we found nonsignificant intergroup difference in BCDVA and a significant difference in UCDVA, in favor of the S-CXL group. This differs from other studies, although we found inconsistent results in several studies. Baenninger et al. and Sarac et al. found nonsignificant difference in the changes between groups for both BCDVA and UCDVA , 25. HowKsteep, Kflat, and Kmean) were also without significant intergroup difference at 12-month follow-up. This is in line with the findings of Sarac et al., who also found no significant difference between groups in simulated keratometry (SimK-1 and SimK-2) after 24 months [Ksteep, Kflat, and Kmean for the S-CXL group.Other keratometric findings . In contrast, Mazzotta et al. in the “Siena Eye-Cross Study 2” reported lower percentage of progression rate (8.33%) in the long-term results after 9 mW A-CXL treatment that occurred within the 2nd and 3rd years. Also, no retreatment was performed after 5 years [In our study, the progression of keratoconus was observed in 18.4% of eyes in the A-CXL group and in 7.1% of eyes in the S-CXL group at 12-month follow-up  at 14 days postoperatively and may disappear after 3 months. A review by Spadea et al.  comparedThis study has several limitations. First, it is a retrospective study. Second, both groups have a small sample size. Hence, a larger study will be required to support these results . ThiThe study's main strength is the utilization and comparison between multiple parameters and intergroup differences. In addition, the surgical method was identical for all patients in this study, apart from the protocol used (S-CXL versus A-CXL).A-CXL is as safe and effective as S-CXL for stabilizing progressive keratoconus in pediatric population. Larger-sample-size studies with a longer follow-up time are required. Considering the long-term results of 9 mW A-CXL and its safety and efficacy profile, it should be preferred to S-CXL for reducing treatment time and improving patients' comfort.


Stiff LA physiology was defined, when the amount of the estimated pulmonary arterial pressure increase between the pre-procedural and the 1-year post-procedural follow-up echocardiography was >10 mmHg and when right ventricular systolic pressure (RVSP) was >35 mmHg at 1-year follow-up echocardiography. The failed rhythm control within 1 year was defined as recurrent AF despite using anti-arrhythmic drugs or cardioversion within a year of AFCA. We explored the incidence and risk factors for stiff LA physiology and the rhythm outcome of AFCA. Among the 1,720 patients, 64 (3.7%) had stiff LA physiology 1 year after AFCA. Stiff LA physiology was independently associated with diabetes , the ratio of the peak mitral flow velocity of the early rapid filling to the early diastolic velocity of the mitral annulus , LA pulse pressure , low LA voltage , empirical extra-pulmonary vein (PV) LA ablation , and radiofrequency (RF) ablation duration . Although the incidence of post-AFCA stiff LA physiology was 3.7% and most of the cases were subclinical, the empirical extra-PV ablation was associated with this undesirable condition. In addition, patients who had low mean LA voltage before AFCA could be susceptible to stiff LA physiology.Catheter ablation is the most effective rhythm control method for patients with atrial fibrillation (AF); however, it inevitably causes atrial tissue damage. We previously reported that AF catheter ablation (AFCA) increases left atrial (LA) pressure without changes in symptom scores. We hypothesized that extensive LA ablation increased the risk of stiff LA physiology. We included 1,720 patients  who underwent  All patients stopped all anti-arrhythmic drugs (AADs) for a period corresponding to at least five half-lives before AFCA.The study protocol adhered to the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of the Yonsei University Health System. All patients provided written informed consent for inclusion in the Yonsei AF Ablation cohort. A total of 1,720 consecutive patients who underwent 2 + right atrial pressure). Stiff LA physiology was defined, when the amount of the estimated increase in PA pressure between the pre-procedural and the 1-year post-procedural follow-up echocardiography was >10 mmHg and when RVSP was >35 mmHg at 1-year follow-up echocardiography  was conducted within 3 months prior to the procedure and at the 1-year follow-up. PA systolic pressure was estimated using RVSP on echocardiography. RVSP was calculated from the peak tricuspid regurgitant jet velocity (V) using the modified Bernoulli's equation  that measured the LA wall thickness by applying Laplace's equation in the cardiac CT images  was calculated using the Law of Laplace [σ = (P × r)/2h ] . We previously reported that the LAW-stress calculated using the abovementioned equation is a useful prognostic parameter for AF recurrence after AFCA   and were compared using the chi-square or Fisher's exact test. The echocardiographic parameters before and 1 year after the procedure were compared using a paired t-test. The logistic regression analysis was used to identify risk factors for stiff LA physiology after AFCA and to estimate the odds ratios (ORs), 95% CIs, and p-values. The variables selected for the multivariate analysis were those with a p-value < 0.05 on the univariate analysis. Since there were variables that had multicollinearity, two logistic regression models were investigated separately. The Kaplan-Meier analysis with log-rank test was used to analyze the probability of freedom from AF recurrences after AFCA. The Statistical Package for the Social Sciences (SPSS) version 25.0 for Windows  and the R software version 3.6.2  were used for the data analysis.Continuous variables were expressed as the mean ± SD for normally distributed variables and as the median with the interquartile range for non-normally distributed variables; they were compared using the Student's de novo AFCA and echocardiography before and 1 year after the procedure were enrolled in this study. We found stiff LA physiology in 64 (3.7%) out of the 1,720 patients 1 year after the de novo procedure (p < 0.001) and had diabetes (p = 0.008), higher proportion of persistent AF (p = 0.001), and higher CHA2DS2-VASc score (p = 0.001) than their counterparts. LA peak pressure (p = 0.005) and LAW-stress (p = 0.001) were higher, and mean LA voltage was lower (p = 0.001) in patients with stiff LA physiology  who underwent rocedure . The patysiology .p < 0.001), LA volume index (p < 0.001), and the ratio of the peak mitral flow velocity of the early rapid filling to the early diastolic velocity of the mitral annulus (E/Em) (p < 0.001) than those without stiff LA physiology at both the pre-procedural and follow-up TTE  and RF ablation (p < 0.001) times and underwent the empirical extra-PV LA ablations more often , E/Em , LA pulse pressure , low LA voltage , empirical extra-PV LA ablation , and RF ablation duration . Furthermore, the low mean LA voltage  and the presence of stiff LA physiology  were independently associated with failed rhythm control within 1 year  rates were significantly higher in patients who had stiff LA physiology 1 year after AFCA (p = 0.002) (During 28 (14.0–56.0) months of follow-up, the clinical recurrence (ter AFCA . On the = 0.002) .de novo AFCA. Stiff LA physiology was independently associated with diabetes, left ventricular (LV) diastolic function, and low LA voltage. Empirical extra-PV ablation was an independent predictor of stiff LA physiology. In addition, rhythm outcomes were worse in patients with stiff LA physiology. Although the incidence of post-AFCA stiff LA physiology was low and most of the cases were subclinical, the empirical extra-PV ablation contributed to this undesirable condition.In this study, we evaluated the incidence and risk factors of stiff LA physiology 1 year after AFCA using echocardiographic estimated RVSP and PA pressure. Stiff LA physiology occurred in 3.7% of patients after p < 0.001, p = 0.001) was also the independent factor of failed rhythm control within a year , and the National Research Foundation of Korea through the Basic Science Research Program (Grant No. NRF-2020R1A2B01001695), which is funded by the Ministry of Science, ICT, and Future Planning.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Starches rich in amylose are promising functional ingredients for calory-reduced foods. In this research, a high-amylose Japonica rice starch (amylose content 33.3%) was esterified with octenyl succinic anhydride (OSA) to improve the functional properties. The OSA-modified derivatives were evaluated for structure and functional properties, with OSA-modified normal Japonica rice starch (amylose content 18.8%) used as control. Fourier transform infrared spectra confirmed the introduction of OSA groups to starch. OSA modification made little change to morphology and particle size of high-amylose starch, but decreased the relative crystallinity and pasting temperature and increased the pasting viscosity, swelling power, emulsifying stability, and resistant starch (RS) content. The changes of properties were related to the degree of substitution (DS). Typically, OSA-modified high-amylose starch at DS of 0.0285 shows polyhedral-shape granules, with a volume-average particle diameter of 8.87 μm, peak viscosity of 5730 cp, and RS content of 35.45%. OSA-modified high-amylose starch had greater peak viscosity and RS content and lower swelling power than OSA-modified normal starch of similar DS, but the two kinds of derivatives did not have a significant difference in emulsifying stability. The OSA-modified high-amylose Japonica rice starch could be used as an emulsifier, thickener, and fat replacer in food systems. Native starches often perform poorly in industrial applications and are tailored through physical or chemical modification to develop desirable functional properties, such as pasting properties, dispersity, and digestibility ,2. OctenThe properties of OSA-modified starch have been affected by many factors, including starch type, amylose content, degree of substitution (DS), etc. Wen et al.  found thOryza sativa L.) is one of the most important cereal crops, providing more than half of the world’s staple food. The Asian cultivated rice grown worldwide is classified into two major subspecies, Indica and Japonica. Indica rice is the major type of rice grown in the tropics and subtropics, for example, central and southern China, India, Pakistan, and Indonesia [Rice , a promising ingredient for calory-reduced food, was chemically modified with OSA. The structure and properties of the OSA-modified high-amylose Japonica rice starch were investigated and compared with OSA-modified normal Japonica rice starch (amylose content 18.8%). This research will be helpful for the design of modified starch with new and enhanced properties.High-amylose Japonica rice (variety Jiangtangdao1) was kindly supplied by the Shanghai Academy of Agricultural Sciences . Normal Japonica rice (varieties of huai5) was obtained from a local grain trader . Soybean oil (Golden Dragon Fish) was supplied by Yihai Kerry Group . Porcine pancreatic-amylase (P7545) and amyloglucosidase (A7095) were purchase from Sigma Chemical Co. . A glucose oxidase/peroxidase (GOPOD) kit was purchased from Megazyme International Ireland Ltd. . Other chemical reagents were of analytical grade unless otherwise stated.Starch was isolated from white rice according to the method of Wang et al. . AccordiStarch  was dispersed in water (140 mL) with stirring. The pH of the slurry was adjusted to 8.5 with 1 mol/L NaOH at 25 °C. OSA  was added to the slurry. The mixture was continuously stirred for reaction, with pH maintained at 8.5. After 6 h of reaction, the starch slurry was neutralized to pH 7.0 with 1 mol/L HCl and then centrifuged at 3000 r/min for 15 min . The residue was washed three times with water and once with acetone, and then dried at 40 °C for 24 h. The degree of substitution (DS) of OSA-modified starch was determined by titration following the method described by Song et al. .w/w) and pressed into transparent tablets under infrared light before measurement. Measurements were made in the region 4000–400 cm−1 at a resolution of 4 cm−1 with a total of 64 scans per spectrum.The Fourier transform infrared (FTIR) spectra of native and OSA-modified starch samples were collected on a Nicolet Nexus 670 FTIR spectrophotometer . Starch samples were mixed with dried KBr powder in a ratio of 1:100  at an accelerating voltage of 15 kV. Before examination, starch samples were sprinkled onto double-sided tape attached to a specimen holder and coated with gold in a vacuum evaporator.The particle size distributions of native and OSA-modified starch samples were measured with the laser light scattering method, by using a Malvern Mastersizer 3000 instrument . Distilled water was used as the suspension medium. Starch samples (100 mg) were dispersed in 5 mL of water and vortexed for 2 min . The dispersion was then swiftly transferred into the sample cell of the particle size analyzer.−1 and a scanning step of 0.033°. The relative crystallinity (%) was quantitatively estimated by using the Origin software  according to the method of Wang et al. [The crystalline structures of native and OSA-modified starch samples were evaluated using an Xpert PRO diffractometer , operating at 40 kW and 40 mA with a Cu Kα radiation source (λ = 0.154 nm). Starch samples were equilibrated to a moisture content of about 10% before the analysis. XRD patterns were acquired for a diffraction angle (2θ) range from 3° to 40° at a scanning speed of 10° ming et al. .The pasting properties of native and OSA-modified starch samples were measured using a Rapid Visco-analyzer  according to the method of Chen et al. . Starch g/g) using the following formula:Swelling power of native and OSA-modified starches were determined by heating starch–water slurries in a water bath at temperatures ranging from 50 to 80 °C in 10 °C intervals, according to the procedures of Wang and Copeland  with somSH is the height of the serum layer, and EH is the total height of the emulsion. A lower creaming index value indicates higher emulsion stability.The emulsifying stability of native and OSA-modified starches was measured according to the method of Lin et al.  with a sIn vitro digestibility of native and OSA-modified starches was analyzed according to the method of Ma et al. . BrieflyData are averages of duplicate observations and expressed as means ± standard deviations. An analysis of variance with a significance level of 5% was conducted, and Duncan’s test was applied to determine differences between means using the commercial statistical package .The DS of OSA-modified normal and high-amylose Japonica rice starches as a response to OSA addition are shown in −1 could be attributed to the vibration of O–H groups. The band at 2942 cm−1 was due to C–H stretching related with ring methane hydrogen atoms. The characteristic peak at 1643 cm−1 originated from the tightly bound water present in the starch. The peaks at 800–1200 cm−1 were due to stretching of the C–O bond.The FTIR spectra of normal and high-amylose Japonica rice starches with and without OSA modification are shown in −1 emerged after OSA modification. This peak was ascribed to the asymmetric stretching vibration of carboxylate RCOO– [−1 was observed in OSA-modified starches, which can be attributed to the characteristic C=O stretching vibration of an ester carbonyl group [−1 than OSA-HighAmyST-1. This result suggests that the increase of DS enhances the ester band absorption of OSA-modified high-amylose Japonica starch.For both normal and high-amylose starches, a new peak at 1571 cmte RCOO– . Anotheryl group . These rp > 0.05). These results suggest that particle size distribution of high-amylose starch was not significantly influenced by the OSA modification.The SEM images of normal and high-amylose Japonica rice starches with and without OSA modification are shown in p < 0.05). The RC of native high-amylose starch was 28.3%, and the derivatives at DS of 0.0177 and 0.0285 were significantly lower at 27.5% and 27.1%, respectively. This was consistent with previous research on OSA modification of indica rice starch and corn starch [XRD patterns of normal and high-amylose Japonica rice starches with and without OSA modification are shown in n starch ,29. Thesp < 0.05). This result was consistent with some previous research on other starches [p < 0.05) after OSA modification, which can be attributed to the weakening of the intermolecular hydrogen bond induced by the introduction of the bulky OSA group [p < 0.05). For OSA-modified high-amylose starch, pasting viscosities generally changed as DS increased. The incorporation of bulky OSA groups enhanced the overall pasting capacity of the starches, and the modified starches tended to paste more extensively [The pasting parameters of normal and high-amylose Japonica rice starches with and without OSA modification are shown in starches ,31,32. Zstarches  reportedSA group . SimilarSA group ,34. For ensively . Differeg/g, while that for native high-amylose starch increased from 2.13 to 7.47 g/g. The increase of amylose and long chains of amylopectin can strengthen the granular structure of starch and inhibit the swelling of granules during heating [p < 0.05). This may be ascribed to the weakening of the intermolecular hydrogen bond due to the introduction of OSA groups [p < 0.05). The two OSA-modified high-amylose starches with different DS had a slight difference in swelling power.The swelling powers of normal and high-amylose Japonica rice starches with and without OSA modification are shown in  heating ,35. AfteA groups . Like nap > 0.05). For normal starch, OSA modification improved the emulsifying stability, as revealed by the decreased creaming indexes of both freshly prepared and stored emulsions (p < 0.05). Compared with emulsions of native high-amylose starch, emulsion of OSA-HighAmyST-1 showed no significant difference in the creaming index of the freshly prepared emulsion (p > 0.05), but showed a slight decrease in the creaming index of the stored emulsion (p < 0.05), suggesting that the emulsifying stability of high-amylose starch was slightly improved after a low degree of OSA modification. The increase of DS enhanced the emulsifying stability of OSA-modified high-amylose starch. For both freshly prepared and stored emulsions, there were no significant differences between the OSA-HighAmyST-2-formulated emulsion and the OSA-NormalST-formulated emulsion in creaming indexes (p > 0.05). This result suggests OSA-modified high-amylose starch had an emulsifying stability comparable to OSA-modified normal starch of similar DS.The creaming indexes of normal and high-amylose Japonica rice starches with and without OSA modification are shown in p < 0.05). This result could be attributed to its higher amylose content and higher amounts of long-length chains (B2 and B3 chains). You et al. [p < 0.05). RS of OSA-modified high-amylose starch increased as DS increased (p < 0.05). Similar results were obtained by Remya et al. [p < 0.05). This result was in agreement with the difference of their native starches in RS content.The RDS, SDS, and RS contents of normal and high-amylose Japonica rice starches with and without OSA modification are shown in u et al.  suggestea et al. , who repOSA-modified high-amylose Japonica rice starch with different DS were prepared. The FTIR spectra confirmed the formation of ester linkage between OSA and starch. Compared with native high-amylose starch, the derivative did not show a significant change in morphology and particle size. OSA modification slightly decreased the relative crystallinity and pasting temperature of high-amylose starch, but increased the pasting viscosity, swelling power, emulsifying stability, and RS content. The changes of these properties were affected by DS. Compared with OSA-modified normal starch of similar DS, the OSA-modified high-amylose starch had a higher peak viscosity and RS content, lower swelling power, and comparable emulsifying stability. The modified high-amylose Japonica rice starch could potentially be employed as emulsifier, thickener, and fat replacer in the food industry, and the application needs further study. Due to the high content of RS, this starch derivative may have many potential health benefits, such as decreasing postprandial glycemic and insulinemic responses, lowering plasma triglyceride concentrations, improving whole-body insulin sensitivity, and reducing fat storage, which also deserve further research attention.


Neopyropia yezoensis, for which genome information has recently been released, the conchocelis apical cell exhibits tip growth and forms a filamentous structure. Here, we report a live-imaging technique using high-resolution microscopy to analyze the tip growth and cell division of N. yezoensis conchocelis. This imaging analysis addressed tip growth dynamics and cell division in conchocelis apical cells. The directionality and tip growth expansion were disrupted by the application of cytoskeletal drugs, suggesting the involvement of microtubules (MTs) and actin filaments (AFs) in these processes. A growing apical cell mostly contained a single chloroplast that moved toward the expanding part of the apical cell. Drug application also inhibited chloroplast movement, implying that the movement may be dependent on the cytoskeleton. The study determined that live-imaging analysis is a versatile approach for exploring the dynamics of tip growth and cell division in N. yezoensis conchocelis, which provides insights into the regulatory mechanisms underlying cellular growth in multicellular red algae.A wide range of tip-growing cells in plants display polarized cell growth, which is an essential cellular process for the form and function of individual cells. Understanding of the regulatory mechanisms underlying tip growth in terrestrial plants has improved. Cellular processes involved in tip growth have also been investigated in some algae species that form filamentous cells, but their regulatory mechanisms remain unclear. In the macro red alga  Arabidopsis thaliana and the moss Physcomitrium patens . This alga, previously referred to as Pyropia yezoensis, is proposed to be reclassified into the new genus Neopyropia based on the molecular phylogenetic analysis  and actin filaments (AFs) may be essential for proper polarized cell expansion and chloroplast movement during tip growth. Moreover, our time-lapse observation of cytokinesis revealed that a chloroplast was separated into two with furrowing of the septum. The usefulness of the live-imaging analysis is addressed to explore the regulatory mechanisms underlying tip growth and cell division in N. yezoensis strain U-51 (–2 s–1 in a long day period (16 h light/8 h dark). The medium was continuously bubbled with filter-sterilized air. For vegetative propagation, the conchocelis cells were collected every week and fragmented with forceps. The fragmented cells were inoculated in fresh (300 mL) medium and incubated under the same condition as described above.For routine subculturing, conchocelis of ain U-51  were culFor microscopic observation, Noriseed was used at a concentration of 0.2% or 0.1% as the nutrient mixture solution.The overall morphology of the conchocelis was observed under a stereo microscopy  equipped with a cooled digital camera .–2 s–1 in a long day period for 7 days, 1.5 mL of the medium was added in the dish, followed by incubation for 7–14 days. The conchocelis attached to a coverslip bottom of the plastic-based dish were observed.For the initial imaging of living cells, conchocelis were cultured in artificial seawater supplemented with the nutrient solution in a glass-based dish  or plastic-based dish  for approximately 2 weeks. For stable imaging, conchocelis were inoculated in 0.5 mL of the medium in a plastic-based dish. After incubation at 20°C under 37 μmol mImage acquisition for time-lapse observation was performed with an Axio observer Z1 inverted microscope with a 40 × /0.85-naumerical aperture (NA) objective lens  or a 63 × /1.40-NA objective lens  equipped with a spinning disc-confocal unit CSU-X1  and a CCD camera  or a CMOS camera .A 488-nm excitation laser was used for autofluorescence of chloroplasts and FM1-43 fluorescence with collection of emission spectrum from 500 to 545-nm. A 561-nm excitation laser was used for autofluorescence of chloroplasts by collecting the 580–640-nm emission spectrum. Chloroplasts contain phycoerythrin that is excited by both 488-nm and 561-nm laser. The fluorescence signal specific to FM1-43 was detected through a comparison between the fluorescence signals with an excitation of 488 nm and 561 nm.Time-lapse observation of chloroplasts during tip growth and cytokinesis was performed at 3-min intervals with Z-scan , using a 561-nm excitation laser. All z-sections were used to perform the maximum Z-projection for analyzing the tip growth. Of the 11 z-sections, five sections were used to process the maximum Z-projection of images of cytokinesis. Time-lapse observation of chloroplasts in the presence of cytoskeletal drugs was performed at 3-min intervals with Z-scan , using a 561-nm excitation laser. Of the 11 z-sections, six sections were used for processing the maximum Z-projection of the images. For long-term imaging of tip-growing conchocelis cells, time-lapse observations were performed using a 561-nm excitation laser. Image acquisition was performed at 20-min intervals with a Z-scan . Four z-sections were used to perform the maximum Z-projection.For time-lapse imaging of FM1-43 fluorescence, image acquisition was performed at 10-min intervals with a Z-scan  or 15-min intervals with a Z-scan . All sections or some selected sections were used to perform the maximum Z-projection. For three-dimensional (3D) images of chloroplasts, z-sections  were collected. For 3D-detection of FM1-43-labeled membranes in the septum, image acquisition was performed at with a Z-scan . All sections were used to perform the maximum Z-projection.For viable staining with Evans blue, an inverted microscopy IX73 (Olympus) with 40 × /0.6-NA objective lens (LUCPLFLN) equipped with a digital camera DP22 (Olympus) was used.http://fiji.sc/Fiji). Chloroplast volume and cell volume was measured by using 3D ImageJ Suite . Conchocelis were cultured in artificial seawater supplemented with 0.2% (v/v) Noriseed for 14 days. After removal of the medium, conchocelis were stained with 2 mL of the medium containing 5 μM FM1-43 and observed in the presence of FM1-43. For tip growth and cytokinesis, image acquisition was performed after the addition of FM1-43. For measurement of the fluorescence intensity of FM1-43 in growing apical domes, image acquisition started from 30 min after the addition of FM1-43 to stain plasma membrane sufficiently.Conchocelis were stained with artificial seawater supplemented with 0.2% (v/v) Noriseed containing 0.05% (w/v) Evans blue  for 10 min. After staining, the cells were washed with the medium five times.–2 s–1 for a long day period. For time-lapse observation, conchocelis were incubated at 20°C for 2 weeks under 37 μmol m–2 s–1 in a long day period in 2.5 mL of the medium using the plastic-based dish as described above. After the medium was removed from the dish, 3 mL of the medium supplemented with 1 μM oryzalin, 10 μM butamifos, or 25 μM latrunculin B was added to the dish, and conchocelis were further cultured for 4 days in the presence of the drugs. When DMSO was used as the solvent, the highest DMSO concentration in the medium was 0.1% (v/v).Oryzalin and butamifos  and latrunculin B  were dissolved in DMSO. After dilution to an appropriate concentration of these drugs, conchocelis cells were incubated in each well of a 24-well plate. After adding 2 mL of the medium containing these drugs into wells, cells were incubated at 20°C for 2 weeks under 37 μmol mTo establish a live imaging system for growing conchocelis cells, dishes and media suitable for imaging living conchocelis cells were determined. Initially, conchocelis cells were cultured in artificial seawater containing 0.2% Noriseed using a glass-based dish and a plastic-based dish for observation with a microscopy. The growth of conchocelis cells in the plastic-based dish was better than that in the glass-based dishes, indicating that the plastic-based dish was appropriate to microscopic imaging . After 1Chloroplasts and vesicles were spatiotemporally organized in the apical dome of tip-growing cells in land plants; chloroplasts and vesicles were visualized by detecting autofluorescence of chloroplasts and fluorescence of intracellular membranes stained with FM1-43, respectively.The Z-projection analysis with z-sections indicated that conchocelis apical cells have a large lobe-shaped chloroplast . The apin = 48, SD = 0.025). During expansion of the apical dome, the protruding part of the chloroplast moved toward the apical expansion zone and was frequently distributed near plasma membrane on the tips of the apical dome  and 54.8% (n = 31) in oryzalin and butamifos, respectively, and 62.5% (n = 72) in latrunculin B] was higher than that in the control treatment [3.2% (n = 62)]. The size of the conchocelis cell clusters under the application of the drugs was smaller than that of the control cell clusters in a dose-dependent fashion  and butamifos (10 μM) for MT disruption, and latrunculin B (25 μM) for AF disruption . The connculin B . Relatival cells . The pro fashion ,6, sugge fashion , 7. Thus fashion . These rn = 5) and 0.24 ± 0.05 μm/min (n = 6) for oryzalin and butamifos, respectively, and 0.19 ± 0.03 μm/min (n = 8) for latrunculin B] was lower than that of the control cells [1.10 ± 0.23 μm/min (n = 13)]. The chloroplasts did not move much in the presence of drugs, indicating that the application of the drugs leads to reduced movement of chloroplasts in the cells.In the presence of the cytoskeletal drugs, the shape of the chloroplasts was also changed. In the growing apical cells in the control condition, the chloroplasts showed a lobed, streamlined shape, but in the presence of oryzalin or latrunculin B, the chloroplasts displayed a wavy mesh shape . ChloropConchocelis tissue is a multicellular filament that is formed through tip growth and successive divisions of a conchocelis apical cell . Thus, wN. yezoensis. Time-lapse imaging analysis addressed the dynamics of polarized expansion at the tips of the apical dome in conchocelis apical cells. The expansion rate of conchocelis apical cells is lower than that of tip-growing cells in land plants, such as pollen tubes and root hairs of the eudicot A. thaliana (Physcomitrium patens (Ectocarpus sp. (In this study, we established a live-imaging technique using high-resolution microscopy to analyze the tip growth of conchocelis cells in thaliana  and caulm patens , and tiprpus sp. . In contrpus sp. . BecauseN. yezoensis are different from those in land plants. In general, the chloroplasts of filamentous algae are present in lower numbers, and chloroplast morphology is variable in algae (N. yezoensis, each vegetative cell of the gametophytic thallus contains one chloroplast in the center (N. yezoensis conchocelis cells indicate that these features are shared in both the sporophyte and gametophyte generation. In the expanding zone of the apical dome, the large chloroplast expanded toward the tips of the apical dome and were close to the apical dome. This distribution of the chloroplast is similar to that in the brown alga Ectocarpus because the chloroplast is distributed in the apical dome in Ectocarpus apical cells (N. yezoensis and the bangiophyte red algae (N. yezoensis.Most conchocelis apical cells have a large, lobed chloroplast that is distributed throughout the cytoplasm of the cells, indicating that the morphology and number of chloroplasts in in algae . In N. ye center . Thus, tal cells . Moreoveal cells . Interesed algae . One posN. yezoensis, kinesin may play a role in promoting the expansion and determining the direction of the growth as a motor protein. Imaging of intracellular membranes stained with FM1-43 revealed that vesicles accumulated in the growing apical dome, suggesting that the vesicles may be targeted at the tips of the apical dome. Vesicle transport may be dependent on kinesin-based activity. The investigation of MT organization and kinesins in the growing apical dome will offer insight into their precise roles in polarized tip growth.Application of cytoskeletal drugs leads to reduced expansion of apical cells and aberrant direction of the conchocelis cells, suggesting that both MTs and AFs are indispensable for proper tip growth of conchocelis apical cells. MTs and AFs are fundamental components that play critical roles in tip growth in land plants . Our resCyanidioschyzon merolae, plastid division and cell division can be highly synchronized (N. yezoensis.Our imaging analysis of cell division in conchocelis apical cells revealed that a large chloroplast was divided through furrowing cytokinesis. This result is consistent with a previous study in another cell type such as spermatogonial cells , indicathronized . The PD During cytokinesis, a single circular region without the FM1-43-labeled intracellular membranes was formed in the center of the septum. In the septum of red algae, pit connections are formed between daughter cells during cytokinesis . A pit cIn conclusion, we have established a live-imaging system for studying tip growth and cell division in the multicellular red alga, which this system could be a useful experimental approach to improve our understanding of the molecular basis of tip growth and cell division in the near future.The original contributions presented in the study are included in the article/YH conceived the experiments. YH, MS, and NT performed the experiments, collected, and analyzed the data. KM contributed to data analysis. YH and KM wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Barbell placement can affect squat performance around the sticking region. This study compared kinematics, kinetics, and myoelectric activity of the safety-bar squat with the high-bar, and low-bar squat around the sticking region. Six recreationally resistance-trained men  and eight women  performed three repetition maximums in all three squat conditions. The participants lifted the least load with the safety bar followed by the high-bar and then the low-bar squat. Greater myoelectric activity of the gluteus maximus was observed during safety-bar squats than high-bar squats. Also, larger knee extension moments were observed for the safety bar compared with low-bar squat. Men had higher myoelectric activity in the safety-bar condition for the gluteus maximus during all regions in comparison with women, and greater knee valgus at the second occurrence of peak barbell velocity. Our findings suggest that the more upright torso inclination during the safety-bar could allow greater gluteus maximus contribution to the hip extensor moment. Moreover, low-bar squats allowed the greatest loads to be lifted, followed by the high-bar and safety-bar squats, possibly due to the larger hip moments and similar knee moments compared to the other squat conditions. Therefore, when the goal is to lift the greatest load possible among recreationally trained men and women, they should first attempt squatting with a low-bar technique, and if the goal is to increase myoelectric activity in the gluteus maximus, a safety-bar squat may be the more effective than the high- bar squat.  The back squat is a commonly used exercise by recreational lifters and athletes, both in training and competition . A back When performing the barbell back squat with high-bar or low-bar barbell placement, studies often analyze lower-body kinematics . Accordi0) to the first located peak velocity (vmax1). The sticking region start from this point to the first located minimum velocity (vmin) in the ascent phase, right before the barbell accelerates again to the next peak velocity of the barbell (vmax2), which is the post sticking region. It is suggested that is there a co-contraction between the quadriceps and the gluteus muscles, which causes a deceleration in the ascent and thereby the sticking region in the back squat [When squatting with loads over 85% of 1-RM, a sticking region occurs during ascent of the squat . The stick squat . This maTo the authors’ best knowledge, safety-bar squats have not previously been compared to high-bar and low-bar squats. Therefore, the purpose of this study was to compare barbell and joint kinematics, kinetics, and myoelectric activity of the safety-bar squat with the high-bar and low-bar squat around the sticking region. We hypothesized that squatting with a safety-bar or high-bar would produce a more vertical torso position due to less peak hip flexion and a forward shift in center of mass, creating greater knee joint moments and thereby, greater myoelectric activity during these squat conditions for the knee extensor muscles. We further hypothesized that the low-bar squat would result in more hip flexion, creating greater hip joint moments and hip extensor myoelectric activity compared to the safety-bar condition.Fourteen participants participated in this study, of whom 6 were men  and 8 were women . Inclusion criteria were: (1) no injury or illness that could reduce maximal squat performance; (2) previously self-reported 1-RM at 1.5 (men) and 1.0 (women) times body mass in the last 12 months; (3) squat depth requirement meeting the International Powerlifting Federation Technical Rules book [0, vmax1, vmin, and vmax2. The dependent variables—myoelectric activity, moment arms, forces, and joint moments—were collected as means during the pre-sticking, sticking, and post-sticking region.To investigate kinematic, kinetic, and myoelectric activity differences around the sticking region in the safety-bar, high-bar and low-bar squat conditions, a within-subjects, repeated measures design was used in which all participants performed all three conditions on one test day after several familiarization days. The dependent variables, barbell velocity, displacement, time, and joint angles, were collected as direct variables in the events vA 3-RM test was used to investigate the different patterns around the sticking region in kinematics, kinetics, and myoelectric activity during a high-bar, low-bar, and safety-bar squat. Three familiarization sessions were conducted before the test session to ensure proper performance during test days and to find the actual 3-RM.www.randomizer.org, accessed on 31 July 2021). During 3-RM familiarization sessions, both mean concentric barbell velocity and the repetitions in a reserve-based rating of perceived exertion [On familiarization day 1, bi-acromion length was measured to decide the stance width for every participant. For this, 0.7 times of the bi-acromion length was used as stance width and standardized during all familiarization sessions and testing. External rotation of the foot was self-selected by the participants during the study but was then standardized and controlled for the rest of the study. The depth requirement was standardized through all familiarizations and test sessions using the rulebook of the International Powerlifting Federation . Grip wiexertion  were meaOn test day, the participants performed a standardized warm-up protocol with the first randomized squat condition. The standardized squat protocol started with taking the lowest obtained familiarization 3-RM for calculating the following: four repetitions at 40%, three repetitions at 55%, and three repetitions at 70% of the lowest obtained familiarization 3-RM. Thereafter, load was increased by 1–10 kg for the randomized squat condition to achieve the recorded mean concentric barbell velocity or if failure occurred on the third repetition. After achieving a 3-RM in a squat condition, the participants tested the next squat condition starting at the lowest obtained familiarization 3-RM in the other squat conditions until true 3-RM were achieved. After all lifts were performed, participants completed a 5-s maximal isometric squat in the bottom position with the same stance widths as the dynamic trials for electromyography (EMG).0), first maximal barbell velocity (vmax1), first located lowest barbell velocity (vmin), and second maximal barbell peak velocity (vmax2). The linear encoder was synchronized with the EMG recordings using a MuscleLab 6000 system and analyzed by MuscleLab version 10.200.90.5095 .A linear encoder  was used to measure the lifting time of the barbell, and the vertical displacement was measured from the lowest point of the barbell with a resolution of 0.019 mm at a 200 Hz sampling rate. The velocity of the barbell was calculated by using the 5-point differential filter with software . The hip, knee, and ankle angles and moments, barbell displacement, and velocity were identified at the following positions during the ascent of the squat during the last repetition: lowest position of the barbell . A preamplifier located as close to the pickup point as possible amplified and filtered the raw EMG signals. The signals were high-pass and low-pass  filtered, rectified, and integrated. Maximal voluntary isometric contraction (MVIC) EMG activity was measured on the participants’ dominant side, placed with SENIAM recommendations  on the f0, vmax1, vmin, and vmax2, four markers were placed on the barbell with a 20 cm distance. To track the 3D ground reaction forces and enable inverse dynamics calculation of joint moments, two force plates  were integrated in the Qualisys motion capture system. The origin of the axes was set to the corner of the left force platform. The x, y, and z axes were set to mediolateral, anterior-posterior, and vertical directions, respectively.A three-dimensional (3D) motion capture system , with eight cameras sampling at a frequency of 500 Hz, was used to track reflective markers, creating a 3D positional measurement. The 3D motion capture system was synchronized with the linear encoder and EMG recordings, using the MuscleLab 6000 system . Markers were placed on both sides of the body, except for the upper and lower hand which were placed on the lateral and medial epicondyle of the humerus and the radial and ulna styloid process. For the thorax, markers were placed on the C7 spinous process of the vertebra, acromion, TV1 thoracal process of the vertebra, the midpoint between the inferior angles of the most caudal points of the two scapulae, and sternum xiphisternal and sternum jugular notch joint . For the0, vmax1, vmin, and vmax2) were analyzed and calculated with a Cardan sequence in the order x-y-z, from distal to proximal orientation. Torso angle was calculated as the angle between the torso segment and the lab, and angles for the hip, knee, and ankle joints were calculated as the angle between the distal and proximal segments , motion capture data were exported as C3D files. A low-pass Butterworth filter at a cut-off frequency of 10 Hz was used for smoothing all calculations from the model-based data. The torso, hip, knee, and ankle, angles in the different events  × 4  × 2 (sexes: men and women) analysis of variance (ANOVA) was used. To assess differences in EMG activity and joint moments between various conditions and regions, a 3 (conditions) × 3 (regions) ANOVA with repeated measures was performed. If significant differences were found, a Holm–Bonferroni post-hoc test was performed. In cases where the sphericity assumption was violated, a Greenhouse–Geisser adjustment for p-values was reported. The significance level was set at p ≤ 0.05. Statistical analysis was performed in SPSS version 21.0 . All results are presented as means ± standard deviations if not otherwise stated. The effect size was evaluated with η2  where 0.01 < η2 < 0.06 constitutes a small effect, a medium effect when 0.06 < η2 < 0.14, and a large effect when η2 > 0.14 [Normality was tested using the Shapiro–Wilk test. To assess differences between sexes in load lifted, an independent samples 2 > 0.14 .p ≤ 0.006, η2 ≥ 0.55). Men lifted significantly more with the high-bar, low-bar, and safety-bar condition compared with women (p ≤ 0.001). The participants lifted significantly more with the low-bar condition compared with the two other squat conditions (p ≤ 0.012). Also, the participants lifted significantly more load with the high-bar condition compared with the safety-bar (p ≤ 0.007). No significant differences were observed between the squat conditions or sexes in velocities, distances, or timing at the ascent phase of the events around the sticking region   .p ≤ 0.023, η2 ≥ 0.31). The low-bar condition had the greatest torso angle followed by the high-bar and safety-bar conditions during all events (p ≤ 0.023) , while in vmin, the low-bar condition had greater hip flexion than the other conditions (p ≤ 0.026). For knee extension angles, a significant effect was found for the events v0 and vmax2 , while no significant effects occurred during vmax1 and vmin . Bonferroni post-hoc tests revealed that the participants squatted with greater knee extension when using the safety-bar condition compared to the low-bar condition at v0 and vmax2 (p ≤ 0.019). A significant effect of squat condition was found for ankle dorsi flexion angle . Bonferroni post-hoc tests revealed that the safety-bar condition had greater ankle dorsiflexion than the low-bar condition in vmax2 (p = 0.004). No other significant difference was found in hip extension, knee extension and ankle dorsi flexion angle for all squat conditions . For knee valgus there was a significant sex difference found at vmax2 in the high-bar and safety-bar conditions  where men had higher knee valgus than women .A significant effect of torso and hip flexion angle was found during all events  . For hipan women . No othep ≤ 0.21, η2 ≤ 0.11). A significant effect of squat condition was found for mean knee moment arms at v0, vmin, and vmax2 . Post-hoc tests showed that at vmin and vmax2 the safety-bar condition had a larger moment arm compared to the low-bar condition (p ≤ 0.006). However, no significant differences were found at v0 and vmax1 for knee moment arms (p ≥ 0.112). Furthermore, a significant difference at vmin was found, with a greater knee moment arm with the high-bar condition compared to safety-bar (p = 0.025). No further significant differences were found between the safety-bar and high-bar conditions for knee moment arms during the different events (p ≥ 0.08). A significant effect of squat condition was found for mean ankle moment arms at v0 and vmin . A post-hoc test showed significant differences in ankle moment arms between the high-bar and low-bar conditions at v0 and vmin (p ≤ 0.032), where the low-bar condition had a greater ankle moment arm than the high-bar.No significant effect of squat condition was found for moment arms in the hip during the different events . Bonferroni post-hoc tests revealed that the hip moment arm decreased significantly from vmin to vmax2 for all squat conditions . Post-hoc tests revealed that the knee moment arm decreased from vmax1 to vmin and vmax2 for all squat conditions (p ≤ 0.001). However, no other significant interactions with events were found for mean hip, knee, or ankle moment arms for any squat conditions .A significant effect of events on the hip moment arm was also found . However, a significant effect of sticking region was found . Bonferroni post-hoc tests revealed that the hip extension moment decreased from region to region for all squat conditions (p ≤ 0.001), while the plantar flexion and knee extension moment decreased from the pre-sticking to the sticking and post-sticking regions (p ≤ 0.014) , but not for the peak knee extension moment . Post-hoc tests showed that the knee extension moment was greater when squatting with a safety-bar condition than with the low-bar condition during both the descent and ascent phases (p ≤ 0.042).No significant effect of squat condition was found for mean or peak hip extension or ankle plantar flexion moments during the ascent  . Furtherp = 0.039, η2 = 0.16). Squatting with a safety-bar led to greater myoelectric activity of the gluteus maximus than squatting with the high-bar technique (p = 0.024), with no differences between the other conditions (p ≥ 0.05). Furthermore, a significant effect of the region was found for myoelectric activity of the erector muscles, gluteus maximus, quadriceps, hamstring, and shank muscles . No significant differences were found in myoelectric activity between the regions for the gluteus medius or adductor longus muscles  (p ≤ 0.02). Furthermore, the gluteus maximus and biceps femoris myoelectric activity increased during each region (p ≤ 0.028), while the semitendinosus increased myoelectric activity from the pre-sticking to the post-sticking region for all three squat conditions. The quadriceps myoelectric activity decreased from the pre-sticking and sticking region to the post-sticking region (p ≤ 0.011), while the gastrocnemius and soleus decreased myoelectric activity from the pre-sticking to the post-sticking region (p ≤ 0.028) , where post-hoc tests revealed a higher gluteus maximus myoelectric activity in the safety-bar condition for men  than women  in all regions (p ≤ 0.04). For the high-bar condition, a significant difference in the post-sticking region was observed, where higher myoelectric activity in the gluteus maximus was observed for men (130.1 ± 19.8%) compared to women (102 ± 40.1) (p = 0.046). No other significant differences were found in myoelectric activity between the sexes for the other muscles in any region during any squat condition .No significant effects were found for squat condition on myoelectric activity except for the gluteus maximus  . Bonferr≤ 0.028) . A signip ≥ 0.09, η2 ≤ 0.19). However, a significant interaction between events was found for force output between the different squat conditions , where post-hoc tests showed that force output decreased from v0 to all other events for all squat conditions (p ≤ 0.001). No other significant differences were found for force output in the other events for any squat condition (p ≥ 0.17).No significant effect of squat condition was found for mean force output at the different events   . The rel0 and vmax1, while the myoelectric activity of the gluteus maximus was lower in the pre-sticking region than in the sticking region and the hip moment decreased in the post-sticking region (min), where the gluteus muscles and hamstrings increases their myoelectric activity. The reason for the gluteus muscles’ low activity at the start of the ascending phase is most likely due to the large muscle length and moment arms, causing a mechanically poor position to exert force (Peak hip moments occurred at vg region . It seemrt force . Also, oThere were two interesting differences found between the sexes. First, there was a difference in myoelectric activity for the gluteus maximus between sexes, in which the gluteus maximus in the safety-bar condition for all regions was higher in activity for men and can be an important variable for the difference in load lifted. Second, there was a higher knee valgus for men compared to women, which can come from differences in certain movement characteristics between the sexes. Between men and women there are some differences in hip anatomy that are worth mentioning, which may help explain differences in knee valgus. For instance, the female pelvis is generally shorter and wider than the male pelvis, which is taller and narrower ,28. AlsoUse of a safety-bar resulted in a more upright torso inclination compared to the high-bar squat, possibly resulting in a more advantageous position for the gluteus maximus to contribute to the hip extensor moment. Moreover, squatting with a low-bar technique allowed for more loads to be lifted, followed by the high-bar and safety-bar squat. This finding may be attributed to the larger hip moments and similar knee moments compared to the other squat conditions. Therefore, we recommend squatting with a low-bar technique when the goal is to lift the most load possible among recreationally trained men and women. Also, our results indicate that when targeting myoelectric activity in the gluteus maximus, squatting with a safety bar may be more effective than the high-bar condition.


A pathological diagnosis of pancreatic cancer should be performed as much as possible to determine the appropriate treatment strategy, but priorities and algorithms for diagnostic methods have not yet been established. In recent years, the endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has become the primary method of collecting tissues from pancreatic disease, but the effect of EUS-FNA on surgical results and prognosis has not been clarified.To evaluate the diagnostic ability of EUS-FNA and its effect on the preoperative diagnosis, surgical outcome, and prognosis of pancreatic cancer.Between January 2005 and June 2017, 293 patients who underwent surgical resection for pancreatic cancer were retrospectively evaluated. The outcomes of interest were the diagnostic ability of EUS-FNA and its influence on the surgical results and prognosis.p < 0.001). The adverse event rate in ERCP was 10.2%, which was significantly higher than EUS-FNA (1.3%) (p = 0.001). Patients were divided into FNA group (N = 160) and non-FNA group (N = 133) for each preoperative diagnostic method. In the study of surgical curability R0 between the two groups, there was no significant difference in FNA group (65.0% [104/160]) and non-FNA group . In the prognostic study, 256 patients with curative R0 or R1 had a recurrence rate was 54.3% (70/129) in the FNA group and 57.4% (73/127) in the non-FNA group. Moreover peritoneal dissemination occurred in 34.3% (24/70) in the FNA group and in 21.9% (16/73) in the non-FNA group, neither of which showed a significant difference. The median survival times of the FNA and non-FNA groups were 955 days and 799 days, respectively, and there was no significant difference between the two groups (log-rank p = 0.735). In the Cox proportional hazards model, factors influencing prognosis, staging, curability, and adjuvant chemotherapy were the dominant factors, but the preoperative diagnostic method (EUS-FNA) itself was not.The diagnostic sensitivity of EUS-FNA was 94.4%, which was significantly higher than that of endoscopic retrograde cholangiopancreatography (ERCP) (45.5%) (EUS-FNA is a safe procedure with a high diagnostic ability for the preoperative examination of pancreatic cancer. It was considered the first choice without the influence of surgical curability, postoperative recurrence, peritoneal dissemination and prognosis. Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States, with 227,000 deaths per year worldwide , 2. SurgEndoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) was first reported by Vilmann et al.  in 1992 This was a multicenter, retrospective cohort study conducted at one academic and two tertiary care centres. The analysis included data on all patients who underwent surgical resection for pancreatic cancer between January 2005 and June 2017. However, patients who met the following criteria were excluded from the analysis: previous history of upper intestinal surgery or any type of malignancy 5 years after the surgery. Written informed consent for endoscopic procedures was obtained from all patients. The consent for participation of patients in this study and its publication was obtained through an opt-out methodology. The study was conducted in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board at each institution and was registered in the UMIN Clinical Trials Registry (UMIN000025993).th Edition, Revised Version) of the Japan Pancreas Society. Adverse Events of the endoscopic procedures were defined according to the lexicon for endoscopic adverse events by the American Society of Gastrointestinal Endoscopy [The patients were divided into two groups; 160 patients who underwent preoperative EUS-FNA (FNA group) and 133 patients who did not (non-FNA group). Survival time was calculated from the day of surgery to the day of death. Operative curability was defined as follows. R0, no evidence of residual tumor; R1, residual tumor in pathological analysis; R2, residual tumor macroscopically. The staging was based on the General Rules for the Study of Pancreatic Cancer  was used for the examinations. In EUS-FNA, the pancreatic lesion was first visualized by EUS, then the needle was advanced into the lesion through the gastric or duodenal wall. For transgastric punctures, a 19G needle was actively used, and for transduodenal punctures, finer needles, such as 22G and 25G, were often used. After removal of the stylet, a syringe was attached to the needle to apply negative pressure (10 ml) followed by to-and-fro movement several times. The specimen was evaluated macroscopically , as we have previously reported . If it wp-value of < 0.20 in the univariate analyses and the diagnostic method (EUS-FNA) were further assessed in the multivariate analysis. A two-sided p-value of < 0.05 was considered to be statistically significant. Statistical analyses were performed using JMP version 10  or R ver. 3.3.1 .The primary outcome of this study was to evaluate the influence of EUS-FNA on the surgical outcomes and prognosis of pancreatic cancer. The secondary outcomes were the diagnostic capability of EUS-FNA for pancreatic tumors and the adverse event rates from endoscopic procedures. Comparisons were performed using Fisher’s exact test or Pearson’s chi-squared test, as appropriate, for categorical variables, and the Mann– Whitney U-test was performed for continuous variables. The Kaplan–Meier method was performed to estimate survival time, and the log-rank test was used for comparing the two groups. A Cox proportional-hazards model was used to estimate hazard ratios for prognosis, and the possible risk factors associated with survival time were also assessed. The following variables were considered to be candidate risk factors: preoperative diagnostic methods (FNA or non-FNA), age, sex, tumor location, tumor diameter, clinical stage, operation curability, and adjuvant chemotherapy. In the analyses, continuous variables were transformed into dichotomous variable with a cut-off value of the median number. The factors with a A total, 293 patients were enrolled this study. The preoperative diagnostic methods of pancreatic cancer included EUS-FNA in 130 patients, ERCP in 58 patients and both EUS-FNA and ERCP in 30 patients. In the remaining 75 patients, pancreatic cancer was diagnosed based only on imaging findings. Furthermore, we classified the patients into the FNA and non-FNA groups. The patient’s breakdown is shown in Fig. p < 0.001). In the EUS-FNA + ERCP combination group (n = 30), only one patient showed an additional effect of ERCP in eight patients who failed to obtain diagnosis with FNA. Adverse events were recognized in 1.3% (2/160) of patients who underwent EUS-FNA and in 10.2% (9/88) for those who underwent ERCP, with significantly lower occurrences in the former (p = 0.001). There was bleeding in one patient, abdominal pain in one patient in EUS-FNA, post-ERCP mild pancreatitis in five patients, post-endoscopic sphincterotomy bleeding in three patients, and bile duct perforation in one patient who underwent ERCP. All adverse events among those who underwent EUS-FNA were successfully managed conservatively, while two patients who underwent ERCP and experienced post-endoscopic sphincterotomy required hemostasis treatment (Table In the EUS-FNA group (N = 160), the median diameter of the long and short axes of the tumor was 23 mm  and 19 mm 5–40 mm), respectively. FNA was performed from the stomach in 57 patients, the duodenal bulb in 60 patients, the second portion of the duodenum in 39 patients, both the stomach and duodenal bulb in three patients, and the duodenal bulb and second portion in one patient. The size of the FNA needles was 19-gauge in 83 patients, 22-gauge in 46 patients, 25-gauge in 25 patients, 20-gauge in four patients, and 21-gauge in two patients. The median number of passes during FNA was 3    in 182 patients, distal pancreatectomy (DP) in 75 patients, total pancreatectomy (TP) in 5 patients, middle pancreatecromy (MP) in one patient, and unresectable in 28 patients. The curability was as follows: R0, 190 patients; R1, 66 patients; R2, 37 patients. The final stage was as follows: I, 21 patients; II, 25 patients; III, 95 patients; IVa, 112 patients; IVb, 40 patients; and postoperative chemotherapy, 80.2% (235/293). There were no significant differences between the FNA and non-FNA groups in the factors that seemed to affect the prognosis of pancreatic cancer, such as surgical curability and adjuvant chemotherapy. In the prognostic study, a total of 256 patients had a curability of R0 or R1, and the recurrence rates were 54.3% (70/129) and 57.4% (73/127) in the FNA and non-FNA groups, respective. Furthermore, peritoneal dissemination occurred in 34.3% (24/70) in the FNA group and 21.9% (16/73) in the non-FNA group, neither of which showed significant differences (Table p < 0.001). The adverse event rates associated with endoscopic procedures were significantly lower at 1.3% (2/160) in EUS-FNA than the 10.2% (9/88) in ERCP-related procedures (p = 0.001). There were no significant difference in the surgical outcomes, surgical curability, and recurrence rate between patients who underwent EUS-FNA (FNA group) and those who did not (non-FNA group). The Kaplan–Meier analysis also confirmed the absence of a significant difference in the overall survival between the two groups (p = 0.735). In the Cox proportional hazards model for overall survival, EUS-FNA was not an independent risk factor.In the present study, we analyzed the preoperative diagnosis of 293 patients with pancreatic cancer. The overall diagnostic sensitivity of EUS-FNA for pancreatic cancer was 94.4%, which was significantly higher than that of ERCP (45.5%) (p < 0.01), respectively. In our study, the overall sensitivity of EUS-FNA and ERCP-related procedures for pancreatic cancer was 94.4% and 45.5% (p < 0.001), respectively. These results suggest that EUS-FNA is a more sensitive preoperative diagnostic method for malignancy during the evaluation of pancreatic masses in comparison with ERCP-related procedures. In addition, recently, new needles with unique tip shapes have been utilized in fine needle biopsy (FNB) and have shown higher diagnostic yield with fewer needle passes [There are two endoscopic approaches for obtaining pathological specimens from pancreatic tumors: the transpapillary approach (ERCP) and the EUS-guided transintestinal approach (EUS-FNA). The transpapillary approach’s sensitivity for a malignant diagnosis is not very high, and endoscopic-related adverse events, such as post-ERCP pancreatitis, are also a concern . On the e passes –27. Preve passes , althouge passes .The overe passes . Considep = 0.21). In this study, three patients had a recurrence in the gastric wall: one (2.6%) patient in the non-FNA group and two patients (1.2%) in the FNA group (p = 0.46) [p = 0.009), and the median overall survival (OS) was 1042 and 557 days, respectively (p = 0.007). The FNA group had better RFS and OS than the non-FNA group because more patients in the FNA group benefited from the chemotherapy administered immediately after surgery [A few articles have reported the long-term outcomes of preoperative EUS-FNA in patients who underwent surgical resection of pancreatic cancer. A retrospective study by Ngamruengphong et al. evaluated 256 patients who underwent pancreatectomy, including 208 patients who underwent EUS-FNA for pancreatic tumors (FNA group) and 48 patients who did not undergo FNA (non-FNA group), with a median follow-up period of 23 months (range 0–111 months). They showed that gastric or peritoneal recurrence occurred in a total of 19 patients: 13 patients (7.7%) in the FNA group vs six patients (15.4%) in the non-FNA group  of 15 cases, EUS-FNA was performed via the gastric body to preferentially diagnose pancreatic body or tail lesions [Although there was no evidence of the influence of preoperative EUS-FNA on the prognosis, needle tract seeding is a matter of concern. There have been several case reports of suspected tumor seeding related to preoperative EUS-FNA. Minaga et al lesions . Yane et lesions .The present study has several limitations. A retrospective study design might have caused biases mainly in patient selection and treatment strategy. ERCP related procedures were more frequently used in the former part of the study, whereas EUS-FNA was more frequently used in the latter part, which might have also caused bias in the clinical outcomes. Since the study included only high-volume centres for both EUS- and ERCP-related procedures, the external validity might be poor, especially for the endoscopic procedure.In conclusion, EUS-FNA was safe and has a high diagnostic ability during the preoperative examination for pancreatic cancer. It was considered the first choice without the influence of surgical curability, postoperative recurrence, peritoneal dissemination, and prognosis. However, since needle tract seeding is observed to have a certain probability, EUS-FNA for resectable tumors, especially those located in the pancreatic body or tail, requires careful consideration of the relationship between risk and benefit. A randomized controlled trial with a multicenter setting is needed to confirm the study results.